Viral infections remain a major medical problem worldwide because of a lack of efficient therapy, prevention or vaccination strategy and because of the rapid development of resistance. Many viruses and virus families causing problematic disorders can be identified. The family of the Flaviviridae (i.e. Dengue virus, HCV, Yellow Fever virus, West Nile virus) can cause major health problems worldwide for mammals including humans. The family of the Herpesviridae includes important human pathogens like Herpes simplex virus (HSV) type 1 and 2 and cause disorders like Herpes Labialis and Herpes Genitalis and so on. Coronaviridae now approximately comprises 15 species, causing in humans respiratory infections (including Severe Acute Respiratory Syndrome (SARS), enteric infections and rarely neurological syndromes).
The World Health Organization estimates that world-wide 170 million people (3% of the world's population) are chronically infected with HCV. These chronic carriers are at risk of developing cirrhosis and/or liver cancer. The only treatment option available today is the use of interferon α-2 (or its pegylated from) either alone or combined with ribavirin. However, sustained response is only observed in about 40% of the patients and treatment is associated with serious adverse effects. There is thus an urgent need for potent and selective inhibitors of the replication of the HCV in order to treat infections with HCV. Also outbreaks of Orthomyxoviruses like influenza, where no treatment exists, create nowadays regularly commotion on a world-wide basis.
HIV (human immunodeficiency virus) is one of the most problematic viral infections with an estimated 40 million people infected worldwide. Currently available drugs for the treatment of HIV include nucleoside reverse transcriptase (RT) inhibitors (i.e. zidovudine, didanosine, stavudine, lamivudine, zalcitabine, abacavir and emtricitabine), the nucleotide RT inhibitor tenofovir, non-nucleoside reverse transcriptase inhibitors (i.e. nevirapine, delavirdine and efavirenz), peptidomimetic protease inhibitors (i.e. saquinavir, indinavir, ritonavir, nelfinavir, amprenavir and lopinavir) and the entry inhibitor enfuvirtide. These compounds are mostly used in combination therapies (HAART) wherein different classes of anti-HIV compounds are combined.
Entry inhibitors are a relatively new class of anti-HIV compounds and the process of HIV entry into host cells provides different targets for the development of antiretroviral drugs. Every step of HIV entry can theoretically be inhibited, namely 1. binding of HIV to the CD4 receptor, 2. binding to coreceptors and 3. fusion of virus and cell.
The envelope protein of HIV is a trimer, with each of the components consisting of 2 subunits, gp41 and gp120. The gp120 subunit of the viral envelope binds to the cellular CD4 molecule; this receptor binding induces conformational changes in the viral envelope protein that include exposure of a previously hidden, highly conserved domain that binds to a second receptor (coreceptor). The viral coreceptors, CCR5 and CXCR4, are members of the chemokine subfamily of 7-transmembrane domain receptors. Coreceptor binding induces conformational changes in the gp41 subunit that result in the insertion of a fusion peptide into the cell membrane and the binding of gp41 helical region 1 and helical region 2, which mechanically draws the viral and cell membranes together and permits membrane fusion.
Enfuvirtide, a fusion inhibitor, is the only entry inhibitor currently approved by the US Food and Drug Administration for use as an antiretroviral agent. Basically, enfuvirtide mimics the structure of helical region 2 of gp41, which binds with helical region 1. By binding with helical region 1, the drug molecule prevents binding to helical region 2 and thus prevents fusion of the viral and cellular membranes. Other not yet marketed HIV-inhibiting entry inhibitors are known in the art and they interact on different levels of the entry process. These include neutralizing monoclonal antibodies directed against the native trimeric structure of the viral envelope; CD4 binding inhibitors, including BMS-806 (which binds in a cleft of gp120 and thus prevents CD4 binding); CCR5 binding inhibitors and CXCR4 binding inhibitors (eg, AMD3100); and fusion inhibitors (eg, the enfuvirtide derivative, T1249).
There exists a variety of carbohydrate-recognizing plant proteins (agglutinins-lectins) that are endowed with anti-HIV activity. The vast majority of carbohydrate-binding plant proteins that show anti-HIV activity are endowed with specificity for α(1-3)- and α(1-6)-mannose (Man) oligomer binding (21-24). Mannose-binding proteins have also been isolated and characterized from prokaryotic organisms such as cyanovirin from the green-blue algae Nostoc ellipsosporum (25,26) and scytovirin from the cyanobacterium Scytonema varium (27). A striking exception among the anti-HIV carbohydrate-binding plant proteins having a different sugar specificity than mannose is represented by UDA, a plant protein derived from the stinging nettle Urtica dioica (22). This plant lectin shows specificity for N-acetylglucosamine (GlcNAc) (28,29). These agents have been shown to inhibit the entry process of the virus, in particular fusion (21). They do not only inhibit HIV infection but also prevent HIV transmission by efficiently blocking cell-to-cell contact. Therefore, the sugar-binding proteins have been suggested as potential microbicide drugs (30), and for the mannose-specific cyanovirin, efficacy to prevent virus infection in Rhesus monkeys has been demonstrated, providing proof of concept (31). It is thought that the carbohydrate-binding plant proteins exert their antiviral action by strongly binding to the sugar moieties present at gp120 of HIV, thereby compromising the required conformational changes in gp120/gp41 for optimal interaction with the (co)-receptors and fusion with the target cell membrane.
Also glycopeptide antibiotics have been described as having an anti-HIV activity and potentially interfering with the entry process of HIV.
One of the major hurdles in HIV therapy is the development of drug resistance that heavily compromises the long-term efficacy of the current (combination) medication.
Also, vaccine development faces huge problems, due to the fact that the immune system fails to efficiently control HIV infection. Antibodies against HIV produced by the humoral immune system act against free virus but may also act against virus-infected cells (1). They bind to the envelope protein gp120 present at the surface of HIV. By doing this, they can directly block virus infection (neutralisation) or may trigger effector systems that lead to viral clearance. The antiviral activity can be mediated by both neutralising and non-neutralising antibodies. Whereas the neutralising antibodies (Nabs) bind to viral proteins that are expressed on the envelope of the free virus particles, non-neutralising antibodies bind to viral proteins mainly expressed on virus-infected cells but not significantly expressed on free virus particles. Generally, neutralising antibodies produced by the humoral immunity are crucial for vaccine-mediated protection against viral diseases. They may act by decreasing the viral efficiency of infection, which is then resolved by the cellular immunity. In fact, neutralisation occurs when a fairly large proportion of available sites on the virion are occupied by antibody, which leads to inhibition of virus attachment to host cells or to interference with the viral entry (fusion) process (1).
However, with the envelope glycoprotein gp120 of HIV being the target of virus-neutralising antibodies, it does not elicit efficient neutralising response in infected people (2). First, little of the envelope surface of primary viruses appears accessible for antibody binding, probably because of oligomerisation of the gp120 proteins and the high degree of glycosylation of the proteins (low antigenicity). Second, the mature carbohydrate oligomers constituting the envelope spikes of HIV appears to stimulate only weak antibody responses (low immunogenicity). Third, intensive viral variation compromises an efficient neutralisation by the immune system (high mutational rate). It was recently shown by Wei et al. (3) that the glycan shield on HIV-1 gp120 (approximately 50% of the gp120 molecule exists of glycans) is evolving during the course of HIV infection in the face of a continuously changing antibody repertoire. Indeed, successive populations of escape virus in patients with acute HIV infection contained mutations In the envelope gene that were unexpectedly sparse and involved primarily changes in N-linked glycosylation sites. These continuous changes in glycan packing efficiently prevent neutralising antibody binding but not receptor binding. In the light of these observations, it could be hypothesized that the abundant glycosylation sites at the surface of the gp120 glycoprotein serve to protect against humoral Immune response against gp120 epitopes critical for HIV infectivity and/or transmission (4). Indeed, carbohydrate regions of glycoproteins are considered as poor immunogens for several reasons, (i) Carbohydrate moieties exhibit microheterogeneity. A same protein sequence exhibits a broad range of glycoforms, causing the deletion of any single antigenic response (5). (ii) Large carbohydrates are flexible and extend considerably from the protein core, being able to cover potential highly immunogenic epitopes (6). (iii) Viruses fully depend on the host glycosylation machinery, and therefore, the glycans attached to viral proteins (potential antigens) are quite similar to those attached to host glycoproteins, resulting in a better tolerance of these carbohydrates (7). Thus, host immunity responses are not very efficient mainly due to the low antigenicity and immunogenicity of the HIV envelope gp120, and the capacity of the virus to efficiently hide highly immunogenic epitopes of its envelope by its glycans. However, strong evidence is available that mutant HIV strains that contain deletions in glycosylation sites of their any trigger the production of specific neutralizing antibodies to previously hidden gp120 epitopes.
As a conclusion, for many pathogenic viral infections and specifically enveloped viruses like HIV, HCV or influenza no efficient treatment is currently available and moreover, the available anti-viral therapies or preventive measures are not sufficient in order to be able to cure, prevent or ameliorate the respective viral infections due to many reasons, like the occurrence of resistance and unfavorable pharmacokinetic or safety profiles. Therefore, there is still a stringent need in the art for potent inhibitors of viruses, more specifically enveloped viruses such as HIV, HCV or influenza. It is the goal of the present invention to satisfy this urgent need by identifying efficient and less harmful treatment or vaccination regimens and pharmaceutically active ingredients and combination of ingredients for the treatment of viral infections in mammals and in humans.